Defective insulin-stimulated glucose uptake is a hallmark of insulin resistance (IR) and type 2 diabetes (T2D). Insulin promotes glucose uptake by triggering the relocation of the glucose transporter GLUT4 from intracellular storage vesicles to the cell surface through exocytosis. To develop effective and safe treatments for IR and T2D, it is crucial to gain a comprehensive understanding of insulin-stimulated GLUT4 exocytosis at the molecular level. GLUT4 exocytosis ? the fusion of GLUT4 vesicles with the plasma membrane ? requires the membrane-anchored SNAREs, the soluble SM proteins, and the C2-domain factor Doc2b. In the previous project period, we reconstituted GLUT4 vesicle fusion in vitro, for the first time, using defined components, which overcame the limitations of conventional approaches and enabled us to attack the problem from a fundamentally new angle. Using this unique reconstitution system, we discovered a stimulatory function of the SM protein Munc18c in SNARE zippering and a membrane-remodeling role of Doc2b in GLUT4 vesicle fusion. In our preliminary studies, we substantially expanded our reconstitution experiments and uncovered new regulatory functions of vesicle fusion proteins. In addition, our CRISPR genetic analyses revealed Munc18b as another SM protein involved in GLUT4 exocytosis. Based on these major advances, we will first define how Munc18b and Munc18c act in concert with SNAREs to drive GLUT4 vesicle fusion using our reconstitution system. Next, we will establish how Doc2b cooperates with Munc18b and Munc18c to control distinct stages of the membrane fusion reaction. We will then validate the findings of the reconstitution studies in adipocytes and muscle cells, using both cultured cell lines and primary tissues isolated from genetically engineered mice. Finally, we will examine whether and how GLUT4 vesicle fusion proteins are altered in insulin-resistant human adipocytes isolated from biopsies of subcutaneous abdominal fat. Completion of this proposed research will fill major gaps in our knowledge of the GLUT4 exocytic pathway. Our findings will also shed light upon the pathogenesis of IR and T2D, and will facilitate the development of novel strategies for therapeutic intervention.